The design and engineering of novel proteins from alternative protein scaffolds has been an emerging field in the last decade with a broad spectrum of applications ranging from structure biology and imaging tools to therapeutic reagents that are currently being tested in the clinic (H K Binz et al., Nat Biotechnol 23, 1257-1268, 2005; H K Binz and A Pluckthun, Curr Opin Biotechnol 16, 459-469, 2005; S S Sidhu and S Koide, Curr Opin Struct Biol 17, 481-487, 2007; A Skerra, Curr Opin Biotechnol 18, 295-304, 2007; C Gronwall and S Stahl, J Biotechnol 140, 254-269, 2009; T Wurch et al., Trends Biotechnol 30, 575-582, 2012; S Banta et al., Annu Rev Biomed Eng 15, 93-113, 2013).
Desirable physical properties of potential alternative scaffold molecules include high thermal stability and reversibility of thermal folding and unfolding. Several methods have been applied to increase the apparent thermal stability of proteins and enzymes, including rational design based on comparison to highly similar thermostable sequences, design of stabilizing disulfide bridges, mutations to increase α-helix propensity, engineering of salt bridges, alteration of the surface charge of the protein, directed evolution, and composition of consensus sequences (Lehmann and Wyss, Curr Opin Biotechnol 12, 371-375, 2001).
Chagasin is an endogenous protein from the parasite Trypanosoma cruzi, which is the causative pathogen of Chagas disease (ACS Monteiro et al., J Cell Sci 114, 3933-3942, 2001). Chagasin is the signature member of the 142 family of cysteine protease inhibitors and was originally discovered in protozoan parasites; proteins homologous to chagasin have been found to be widely distributed in prokaryotes and eukaryotes as well (D J Rigden et al., FEBS Lett 504, 41-44, 2001; S J Sanderson et al., FEBS Lett 542, 12-16, 2003). Chagasin was isolated as a heat-stable protein that binds to the major lysosomal cysteine protease cruzipain that is essential for the parasite to invade and multiply in mammalian host cells. Besides cruzipain, chagasin also inhibits other cysteine proteases such as Falcipain 2, Falcipain 3, Cathepsins B, L, K and H (S X Wang et al., Structure 15, 535-543, 2007). To date, several homologs of chagasin have been described (D Salmon et al., J Mol Biol 357, 1511-1521, 2006; B O Smith et al., J Biol Chem 281, 5821-5828, 2006; G Hansen et al., Structure 19, 919-929, 2011). Crystal structures of chagasin alone and in complex with the cysteine proteases papain, cathepsin B, cathepsin L and Falcipain2 have shown that three loops (designated L2, L4 and L6 or BC, DE and FG, respectively) protruding out from the β-sandwich protein scaffold are essential for binding to the active site cleft of these proteases in order to inhibit enzymatic activity (A A Figueiredo da Silva et al., J Struct Biol 157, 416-423, 2007; A Ljunggren et al., J Mol Biol 371, 137-153, 2007; S X Wang et al., Structure 15, 535-543, 2007; I Redzynia et al., J Biol Chem 283, 22815-22825, 2008; I Redzynia et al., FEBS J 276, 793-806, 2009). Chagasin has a unique immunoglobulin-like fold with homology to human CD8α (DJ Rigden et al., FEBS Lett 504, 41-44, 2001; S X Wang et al., Structure 15, 535-543, 2007).
Thus, there is a need to develop small, stable, artificial antibody-like molecules for a variety of therapeutic and diagnostic applications. The present invention meets this and other needs.